REQUIREMENT OF GAMMA-CARBOXYGLUTAMIC ACID RESIDUES FOR THE BIOLOGICAL-ACTIVITY OF GAS6 - CONTRIBUTION OF ENDOGENOUS GAS6 TO THE PROLIFERATION OF VASCULAR SMOOTH-MUSCLE CELLS

Gas6 (encoded by growth-arrest-specific gene 6) is a gamma-carboxy-glu tamic acid (Gla)-containing protein which is released from growth-arre sted vascular smooth muscle cells (VSMCs) and potentiates VSMC prolife ration induced by Ca2+-mobilizing growth factors, but not that induced by receptor tyrosine kinases. In this study we examined the importanc e of Gla residues for the biological activities of Gas6 and tried to a ssess the importance of endogenous Gas6 in VSMC proliferation. We demo nstrated that Gla-deficient Gas6 lacked receptor-binding and growth-po tentiating activities. Therefore the vitamin K-dependent modification of Gas6 appeared to be essential for its biological activities. Next w e used warfarin, an inhibitor of vitamin K-dependent gamma-carboxylati on, to estimate the contribution of endogenous Gas6 to VSMC proliferat ion. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Theref ore the inhibition seems to be due to prevention of the vitamin K-depe ndent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing antibody against Gas6 gave a similar result, i.e. it inhibited thrombin-induced VSMC pr oliferation but not that induced by epidermal growth factor. These res ults indicate that endogenously produced Gas6 is very important for VS MC proliferation induced by Ca2+-mobilizing growth factors dependent g amma-carboxylation, to estimate the contribution of endogenous Gas6 to VSMC proliferation. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Therefore the inhibition seems to be due to prevention of th e vitamin K-dependent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing a ntibody against Gas6 gave a similar result, i.e. it inhibited thrombin -induced VSMC proliferation but not that induced by epidermal growth f actor. These results indicate that endogenously produced Gas6 is very important for VSMC proliferation induced by Ca2+-mobilizing growth fac tors.